Inflammation is an early and important response to tissue injury and infection. In the absence of pathogens, it is considered sterile and exacerbates the pathology of major non-communicable diseases (e.g. stroke, myocardial infarction, Alzheimer's, atherosclerosis, diabetes, cancer)1,2. Inflammatory cytokines associated with sterile inflammation are members of the interleukin-1 (IL-1) family, namely IL-1α and IL-1β1. On-going research is establishing the NLRP3-inflammasome complex as one of the most important regulators of inflammation. The NLRP3 inflammasome forms a molecular platform inside macrophages and microglial cells, catalysing the activation of the protease caspase-1. Caspase-1 is responsible for converting the potent pro-inflammatory cytokine interleukin-1 beta (IL-1β) from an inactive to an active secreted form. The NLRP3 inflammasome represents an important new target for the development of novel therapeutics. We have developed novel small molecule inhibitors of the NLRP3 inflammasome and have characterised their potency in vitro.
IL-1β is produced as a pro-IL-1β precursor. This precursor is expressed in response to pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) that bind to pattern recognition receptors (PRRs) on macrophages to upregulate pro-inflammatory gene expression3,4. PAMPs are motifs carried by pathogens, such as bacterial endotoxin (or lipopolysaccharide (LPS)), and DAMPs are commonly endogenous molecules released by necrosis. Pro-IL-1β is inactive and remains intracellular until a further PAMP or DAMP stimulation activates cytosolic PRRs, often of the NLR family, to form large multi-protein complexes called inflammasomes5. These complexes consist of the PRR, pro-caspase-1, and, depending upon the PRR, an adaptor protein called ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), that interact via CARD and pyrin homology binding domains5. When the PRR senses PAMPs or DAMPs it recruits ASC, which in turn recruits caspase-1 causing its activation (FIG. 1). Caspase-1 then processes pro-IL-1β to a mature form that is rapidly secreted from the cell5. Although the closely related pro-IL-1α is not a substrate for caspase-1, the activation of inflammasomes can also drive IL-1α release6. The activation of caspase-1 itself can also cause cell death directly7.
A number of inflammasome forming PRRs have been identified including NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, IFI16, and RIG-I5. Of these inflammasomes identified to-date, the best characterised, and most strongly associated with sterile inflammation is formed by NLRP3.
There are many endogenous (‘sterile’) activators of NLRP3 including molecules such as ATP8, sphingosine9, crystals of monosodium urate (MSU)10, cholesterol11, and Aβ fibrils12. There is limited evidence supporting a direct interaction between DAMP and NLRP3 and additional host cellular signals such as K+ efflux13, lysosomal destabilisation and cathepsin activity14, ROS production and/or mitochondrial dysfunction15, post-translational modification such as deubiquitination of inflammasome components16-16, are suggested to be important.
Anti-IL-1β therapies (such as Anakinra) are remarkably safe, with far fewer incidences of opportunistic infections compared with anti-TNF therapies19. Furthermore, selectively targeting the NLRP3 inflammasome will avoid major anti-microbial inflammasomes such as NLRC4 or AIM220 and are thus even less likely to cause immunosuppressive effects.
The present invention was devised with the foregoing in mind.